BBa_K1911001: pLac ClpXP CI

pLac ClpXP CI design

This year, Lambert iGEM improved upon the characterization of pLac ClpXP CI (part number BBa_K1911001), a part submitted by Lambert’s 2016 iGEM team. pLac ClpXP CI is the third part of their inducible construct and codes for ClpXP, a non-lysosomal protein degradation system, and CI, a competitive inhibitor that, once expressed, inhibits further transcription of p-lambda-rLacI tsPurple. Last year, there was an error in the submitted sequence where the beginning of the construct was omitted from the parts registry page. This year’s team recognized the problem and fully sequenced pLac ClpXP CI to determine the correct sequence. They began by sequencing with VF2 and VR, however since pLac ClpXP CI is almost 3000 base pairs long, the initial sequencing did not capture the middle part of the sequence. To fix this problem, primers were designed to capture the middle thousand base pairs; the results of this sequencing confirmed the middle part of the construct. After comparing this year’s sequencing results to last year’s submitted part, the team realized there was a double terminator at the beginning of the sequence, which would stop transcription from pλR LacI tsPurple, the earlier part of the construct. In light of this information, the parts registry page from last year was updated for clarity on the full construct design.